A Comprehensive Study of Tumor Necrosis Factor-Alpha Genetic Polymorphisms, its Expression in Skin and Relation to Histopathological Features in Psoriasis.

BACKGROUND: Tumor necrosis factor-alpha (TNFα) is an important inflammatory mediator in psoriasis and several genetic polymorphisms of this cytokine have been reported. Majority of studies have focused on the increased G- A polymorphism at the -308 position in psoriasis. There has been no comprehensive study evaluating the genetic polymorphisms, TNFα expression in the skin and histopathology. We are undertaking this study to outline TNFα genetic polymorphisms, its skin expression and histopathological correlation to help determine its role at the genetic and protein level.
MATERIALS AND METHODS: 112 patients of psoriasis and 243 healthy controls were included in this prospective study. 5 ml of peripheral blood was collected to study the TNFα genetic polymorphisms by polymerase chain reaction and restriction fragment length polymorphism analysis. Histopathological analysis of biopsies from the 112 patients were done using visual analogue scale and correlated with the findings. 61 of these cases were analyzed for TNFα expression by immunohistochemistry. The results of study were statistically analyzed using SPSS 13.0 statistical package program.
RESULTS: A strong association of TNFα -308 G/A polymorphism in psoriasis cases was detected. The A allele of the TNFα -308 G/A polymorphism occurs rarely in the Indian population, however there is an over representation of this allele in psoriatic patients. There was no association seen between TNFα genotype and histopathological severity of psoriasis.
CONCLUSION: The study emphasized the central role of TNFα in the pathogenesis of psoriasis. TNFα genotyping may be helpful in identifying subjects in whom anti-TNFα therapeutic strategies may be tried.

What was known?

TNFα is an important inflammatory mediator in psoriasis.

Cytokine gene polymorphisms have been found to alter gene transcription thereby influencing inflammatory processes.

TNFα gene polymorphisms have a role to play in pathogenesis of psoriasis.

Introduction

Psoriasis is a chronic inflammatory disease affecting about 1-3% of Caucasian population.[1] TNFα is an important inflammatory mediator in psoriasis and its expression has been shown to be increased in psoriatic lesions.[2] Cytokine gene polymorphisms have recently attracted considerable interest as they have been found to alter gene transcription thereby influencing inflammatory processes in response to various diseases.[34]

Several polymorphisms have been reported in the TNFα promoter, most of which are functionally silent. Majority of the studies have focused on the G–A polymorphism at the –308 position which was originally misnumbered and known as TNFα –307 [Level of evidence: IIb].[5]

Studies have evaluated TNFα gene polymorphisms in psoriatic patients.[67] However, there has been no comprehensive study evaluating the genetic polymorphisms, TNFα expression in the skin and histopathology. In this study, we evaluated the association between TNFα polymorphism at position –308 and predisposition to psoriasis vulgaris, relationship of polymorphisms with the levels of TNFα in different locations in the skin and finally, relationship of genetic polymorphisms with histopathology. This would help in better understanding of the role of TNFα, at the genetic and protein level.

Materials and Methods

Patients and controls

This prospective study included 112 patients with newly diagnosed psoriasis vulgaris at a large teaching institute. The patients were drawn from the state of Maharashtra, India with no distinction made for religion, caste or tribe. The genetic polymorphisms of these patients were compared with 243 healthy blood donors (controls) who were age and sex matched. 5 ml of peripheral blood was collected from both groups to study the TNFα genetic polymorphisms. The study was conducted in accordance with principles of the Helsinki Declaration and was approved by the institutional ethics committee.

Histopathology

Biopsies were taken from the lesion of patients to include a small portion of the perilesional skin. The biopsies were fixed in formalin, sections were stained with H and E stain and histopathological analysis was done which included seven criteria (epithelial hyperplasia, parakeratosis, Munro's and Kogoj's microabscesses, suprapapillary thinning, inflammatory infiltrate and widened rete ridges). Grading was done using a visual analogue scale and the biopsies were graded as 0 to 3 (Nil to marked).

Immunohistochemistry

61 cases were analyzed for TNFα expression by immunohistochemistry. Serial sections were mounted on poly-l lysine-coated slides. Paraffin sections were immersed in xylene for 5 minutes and hydrated using a gradient series of alcohol. Antigen retrieval was routinely performed by immersing the sections in citric acid buffer (pH 6.0) in a microwave oven for 15 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min and then incubated with a primary antibody in a humidified chamber at 4 overnight. Primary antibody was monoclonal mouse anti-TNFα antibody (Santa Cruz Biotechnology Inc, Santa Cruz, California, USA) at 1:200 dilution. A total of 500 keratinocytes were enumerated in an area that was stained intensely on each three serial slides at 400x magnification and an average was taken. The conventional technique of grid (10 × 10 mm with 1-mm2 boxes) within the eye piece of the microscope was used to analyze the percentage of positive cells. Localization of immunohistochemical staining was grouped and classified as epidermal, basal cell and monocyte staining.

DNA extraction and genotyping of cytokine gene polymorphisms

Genomic DNA was retrieved from peripheral blood by using a standard kit (Quiagen). TNFα gene polymorphisms were genotyped by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. For RFLP analysis of the TNFα –308 polymorphism, the region containing the polymorphic site was amplified using the primer sequence as noted in Table 1. PCR conditions were as follows: 95°C for 5 min, then 35 cycles of 94°C for 30s, 56°C for 30s, 72°C for 30s and a final extension of 72°C for 5 minutes. The products were digested with 10 U of Nco1 at 37°° C for 15 minutes. Fragments were analyzed by electrophoresis on 15% polyacrylamide gel and stained with ethidium bromide. This yielded products of 107 bp (AA allele), 87 bp and 20 bp (GG allele) and 107, 87 and 20 bp (AG allele). The A allele was considered pro inflammatory. All PCR reactions were performed in an Eppendorf Thermal Cycler (MJ Research Inc., Waltham MA).

Table 1

The primer sequences, site of polymorphism and the restriction enzyme used in the RFLP

Statistical analysis

The results of study were statistically analyzed using statistical package program, Statistical Package for Social Sciences (SPSS) 13.0, SPSS Inc, 233 South Wacker Drive, 11th Floor, Chicago, IL, 60606-6412, USA. The descriptive data were given as mean and standard deviation. The Chi-square test was used for comparing the differences between groups. A value of P < 0.05 was considered to indicate statistical significance.

Results

Demographic variables

112 cases (224 alleles) of psoriasis were analysed in the present study. The mean age was 38.9 years with a range of 13 to 76 years. 63 males and 49 females comprised the study population.

TNFα –308 A/G polymorphism Association of Genetic Polymorphisms – Cases vs Controls

It is to be noted that every patient and control has two alleles each which would amount to double the alleles as the actual number of patients and controls. The A allele and AG genotype was more common in the cases as opposed to the controls.

In controls, all the frequencies of tested genotypes and alleles were in Hardy–Weinberg equilibrium. There was a strong association of TNFα –308 G/A polymorphism with respect to both genotypes and alleles in patient group due to the significant difference between patients and controls. Frequencies of genotypes and alleles were –308 GG genotype: 163/243 (67.07%) versus 7/112 (6.25%); GA genotype: 71/243 (29.12%) versus 99/112 (88.39%); AA genotype: 9/243 (3.7%) versus 6/112 (5.35%); –308 G allele: 397/486 (81.68%) versus 113/224 (50.44%) and A allele: 89/486 (18.32%) versus 111/224 (49.55%) between controls and patients, respectively [Table 2].

Table 2

Frequency of TNFα Genotypes and allelic polymorphisms among psoriasis

Evaluation of TNFα immunostaining

TNFα immunostain was evaluated in different compartments of the skin. The immunostain was graded in the form of a percentage i.e. the number of cells showing TNFα immunopositivity as compared to total number of cells. A total of 500 cells were counted and they constituted the denominator. Basal layer, rest of epidermis and monocytes present in upper dermis were compartmentalized.

Since immunostaining was done in 61 cases, relationships of the genetic polymorphisms were analyzed separately in these cases. Of the 61 cases analyzed, 53 showed an AG genotype. The GG and AA genotype was seen in four cases each.

Epidermal positivity for TNFα and correlation with genetic polymorphisms

The percentage of epidermal cells showing cytoplasmic staining for TNFα ranged from 0 to 100% (average: 16.95%) [Figure 1]. There was a weak correlation between the TNFα genotypes and the grade of epidermal immunostaining (P = 0.041).

Figure 1

The TNFα immunostain in the cytoplasm of the epidermal cells. (a) The figure on the left shows a strong positive TNFα immunostain. (b) The figure on the right is a negative control and shows most of the epidermal cells have not stained for TNFα [×40]

Basal cell staining

The percentage of basal cells showing a nuclear staining for TNFα ranged from 0 to 100 (average: 37.95%). There was no correlation between the genetic polymorphisms and the grade of basal cell staining (P = 0.66).

Monocyte staining

The percentage of monocytes showing a cytoplasmic staining for TNFα ranged from 2 to 100 (average: 38.39%). There was no correlation between the TNFα genotypes and the grade of monocyte immunostaining (P = 0.672). The correlation between genetic polymorphism of TNFα with epidermal, basal cell and monocyte staining are as depicted in Figure 2.

Figure 2

Box plots showing correlation between the TNFα genotypes and the grade of epidermal cytoplasmic staining, basal cell staining and monocyte staining of TNFα, respectively

Association of gene polymorphisms with histological features

Biopsies taken from the lesional skin were evaluated using seven criteria as outlined previously. Of the seven, epidermal hyperplasia, inflammatory infiltrate and capillary dilatation were considered the most relevant in the pathogenesis of psoriasis. There was no association seen between the TNFα genotype and the histopathological severity of psoriasis [Table 3 and Figure 3].

Table 3

The association between the TNFα genotypes and the histopathological indicators of severity

Figure 3

Box plots showing the lack of a relationship between TNFα polymorphisms and the inflammatory infiltrate, epidermal hyperplasia, suprapapillary thinning and capillary dilatation

Discussion

Ethnic variability and differences in allele/genotype frequency distribution among different ethnic population is considered to be the key factor in association studies. TNFα –308 A allele has a prevalence of 7% in China and Korea,[89] 15% in Germany,[10] and 11% in normal Italian subjects.[1112] However, the Portuguese,[12] Finnish,[13] and US population,[14] have 13%, 14% and 15% frequencies of –308A allele respectively [Level of evidence: Ib]. In India, TNFα –308 A allele frequency falls between the values for China/Korea and Italy [Level of evidence: III].

Our study has shown that the TNFα AG genotype is seen more commonly in cases of psoriasis as opposed to the controls. Different studies have shown contradictory findings in relation to TNFα genetic polymorphisms and psoriasis. In a study involving 78 patients and 74 healthy controls, Baran et al. found no association between TNFα promoter polymorphism (–308) and psoriasis vulgaris in a Polish population [Level of evidence: IIa].[15] Similar studies have been done in the Korean and Japanese populations and these studies did not show an association between the different genotypes [Level of evidence: IIa].[1617] These studies reported that the most commonly observed genotype (both in patients and control group) was GG (low TNFα producer) and that AA homozygosity (high TNFα producer) was only accidentally present in population. Our findings are in consonance with the fact that the AA genotype is only present accidentally in the population (5.3% of cases and 3.7% of controls). However, in our study we found a large number of patients with the AG genotype. This suggests that in the Indian population at least, the A allele is overrepresented in the cases of psoriasis.

In a meta analysis performed on 1156 psoriasis cases and 1083 control subjects from 10 published case–control studies for the –308 G/A polymorphism, it was found that there was a significantly reduced psoriasis risk associated with the variant GA/AA genotypes of the –308 G/A compared with the GG genotype (OR: 0.57, 95% CI: 0.45–0.71) [Level of evidence: Level Ia].[18] The present study contradicts these findings. We suggest that in the Indian population, the A allele of the TNFα –308 G/A polymorphism occurs rarely; more often seen as an accident. However, there is an over representation in cases of psoriasis. We did not come across any Indian study which has studied this genetic polymorphism in psoriasis; however, we do have circumstantial evidence. In a study evaluating TNFα –308 G/A polymorphisms in gastritis, Achyut et al. have shown that this polymorphism shows an increasing propensity to be associated with the formation of lymphoid-follicles (odds ratio = 2.8) which are precursors to MALTomas [Level of evidence: III].[19] The role of the functional status of the TNFα –308 G/A polymorphism has been debated.[2021] However, in Indians, it has been shown that the TNFα –308 GA genotype was associated with significantly higher production of TNFα as compared to GG genotype [Level of evidence: IIb].[22] These two findings combined with the fact that anti TNFα inhibitors are very successful in the treatment of psoriasis in Indians,[23] suggests the following: (i) significantly associated with psoriatic patients suggests that there is an increased production of TNFα in psoriatic plaques in Indians.

Our second finding showing a correlation between the TNFα –308 G/A polymorphism and the grade of TNFα immunostaining in the epidermis is an extension of the above findings. The interaction between cytokines, especially TNFα and growth factors contributes to the hyperproliferation of keratinocytes, increased neovascularization and inflammation that is frequently observed in psoriasis vulgaris.[24] Our finding that the TNFα gene polymorphisms correlate with the epidermal cytoplasmic expression of TNFα further reiterates the importance of TNFα –308 G/A polymorphism in the pathogenesis of psoriasis.

The other interesting finding was that there was no association between the genetic polymorphisms and the histopathological indicators of severity. In a similar study done in cases of gastritis, there was no correlation between the TNFα –308 G/A polymorphism and the histopathological severity of gastritis [Level of evidence.[25] This finding must be interpreted in the light of the fact that genetic polymorphisms need not necessarily correlate with the final pathology. Genetic polymorphisms may influence the rate of gene transcription, the stability of the messenger RNA or the quantity and activity of the resulting protein. It is the final protein product like TNFα which influences the histology of the lesion. Given the long pathway involved in the final pathogenesis, it is perhaps not surprising that the gene polymorphisms do not correlate with the histopathology.

These genetic polymorphisms may help us to predict response to therapy. Polymorphisms in certain genes like TNF receptor superfamily 1B (TNFR1B) and TNFα-induced protein 3 gene (TNFAIP3) have been found to be associated with response to anti-TNF therapy in psoriatic patients. It would enable the creation of a gene bank, study of the underlying pathogenic mechanisms of psoriasis, identification of other possible therapeutic targets, correlation of results with treatment response and toxicity and increase our knowledge of mechanism of action at molecular level of various drugs and personalise treatment to each individual patient.

In conclusion, the TNFα –308 G/A polymorphism is significantly associated with psoriasis. The TNFα –308 G/A polymorphism is also associated with a heightened secretion of TNFα in the cytoplasm of the epidermal cells. The lack of correlation with the histopathological parameters of severity has been explained. The study emphasised the central role of TNFα in the pathogenesis of psoriasis; TNFα genotyping in patients with psoriasis may be potentially helpful in identifying subjects in whom anti TNFα therapeutic strategies may be tried.

What is new?

TNFα –308 G/A polymorphism is significantly associated with psoriasis.

The lack of correlation of TNFα genetic polymorphisms with histopathological parameters means that other factors also have an important role to play.